Insect repellent compounds

ABSTRACT

Dihydronepetalactone, a minor natural constituent of the essential oil of catmints ( Nepeta  spp.) such as  Nepeta cataria , has been identified as an effective insect repellent compound. Synthesis of dihydronepetalactone may be achieved by hydrogenation of nepetalactone, the major constituent of catmint essential oils. This compound, which also has fragrance properties, may be used commercially for its insect repellent properties.

FIELD OF THE INVENTION

This application claims the benefit of U.S. Provisional Application No.60/366,147, filed Mar. 20, 2002, which is incorporated in its entiretyas a part hereof for all purposes.

FIELD OF THE INVENTION

The present invention relates to the field of insect repellency, and theuse of dihydronepetalactone stereoisomers generally as repellentmaterials.

BACKGROUND OF THE INVENTION

Repellent substances generally cause insects to be driven away from, orto reject, otherwise insect-acceptable food sources or habitats. Mostknown repellents are only mildly toxic. A few of the known repellents,in fact, are not active poisons at all but rather prevent damage toplants/animals or articles of manufacture by making insect food sourcesor living conditions unattractive or offensive. Most current commercialinsect repellents contain the synthetic chemical N,N-diethyl-m-toluamide(DEET) as their primary active ingredient. For instance, repellents soldunder the major commercial brand names such as Off!®, Deep Woods Off!®,and Cutter® are all DEET based products and comprise 85% of insectrepellent sales (Consumer Reports Buying Guide, 1994 Special Year-EndIssue). Further, Consumer Reports tests indicated that products with thehighest concentration of DEET lasted the longest against mosquitoes.Despite being an effective repellent, however, this compound has certaindrawbacks. Specifically, it possesses an unpleasant odor and imparts agreasy feel to the skin. Although it has recently been re-registered forthe use in the US by the EPA, concerns have been raised as to itssafety, particularly when applied to children (Briassoulis, G,;Narlioglou, M.; Hatzis, T. (2001) Human & Experimental Toxicology 20(1),8-14). Studies have demonstrated that high concentrations of DEET maygive rise to allergic or toxic reactions in some individuals. Otherdisadvantages associated with DEET include: 1) it is a syntheticchemical having a limited spectrum of activity; 2) DEET is a powerfulplasticizer and will dissolve or mar many plastics and painted surfaces;and 3) DEET plasticizes the inert ingredients typically used in topicalformulations in order to lengthen the time of effectiveness. This leadsto DEET formulations with low user acceptability.

As a result of the above limitations, DEET-free products with repellentactivity are finding favor with consumers, and demand for compositionscontaining natural products (versus synthetic chemicals such as BEET) isincreasing. These DEET-free repellent compounds require a combination ofexcellent repellency, high residual activity and relatively little or notoxicity to humans (or pets) and the environment. In response to theseconsumer demands, there is an on-going need to develop new repellentcompounds, which can be obtained from, or synthesized from, naturalplant materials and which are pleasant to use.

Many plant species produce essential oils (aromatic oils) which are usedas natural sources of insect repellent and fragrant chemicals [Hay, R.K. M., Svoboda, K. P., Botany, in ‘Volatile Oil Crops: their biology,chemistry and production’. Hay, R. K. M., Waterman, P. G. (eds.).Longman Group UK Limited (1993)]. Citronella oil, known for its generalrepellence towards insects, is obtained from the graminaceous plantsCymbopogon winterianus and C. nardus. Examples of plants used as sourcesof fragrant chemicals include Melissa officinalis (Melissa), Perillafrutescens (Perilla), Posostemon cablin (Patchouli) and variousLavandula spp. (Lavender). All of these examples of plants yielding oilof value to the fragrance industry are members of the Labiatae(Lamiaceae) family. Plants of the genus Nepeta (catmints) are alsomembers of this family, and produce an essential oil, which is a minoritem of commerce. This oil is very rich in a class of monoterpenoidcompounds known as iridoids [Inouye, H. Iridoids. Methods in PlantBiochemistry 7:99-143 (1991)], more specifically themethylcyclopentanoid nepetalactones [Clark, L. J. et al. The PlantJournal, 11:1387-1393 (1997)] and derivatives.

Iridoid monoterpenoids have long been known to be effective repellentsto a variety of insect species (Eisner, T. Science 146:1318-1320 (1964);Eisner, T. Science 148:966-968 (1965); Peterson, C. and J. Coats,Pesticide Outlook 12:154-158 (2001); and Peterson, C. et al. Abstractsof Papers American Chemical Society, (2001) 222 (1-2): AGRO73). U.S.Pat. No. 4,663,346 discloses insect repellants with compositionscontaining bicyclic iridoid lactones (e.g., iridomyrmecin). Further,U.S. Pat. No. 4,869,896 discloses use of these bicyclic iridoid lactonecompositions in potentiated insect repellent mixtures with DEET.

Formal studies concerning the repellency of dihydronepetalactones, aclass of iridoid monoterpenoids derived from nepetalactones (shown inFIG. 1), have been much less conclusive and have failed to teach orimply that these compounds exert a repellent effect on the common insectpests of human society. For example, a study of the composition of thesecretion from anal glands of the ant Iridomyrmex nitidus showed thatisodihydronepetalactone was present in appreciable amounts, togetherwith isoiridomyrmecin (Cavill, G. W. K., and D. V. Clark. J. InsectPhysiol. 13:131-135 (1967)). Although isoiridomyrmecin was known at thetime to possess good ‘knockdown’ insecticidal activity, no evidence wasprovided in support of a similar activity for isodihydronepetalactone,and no investigation of this compound's repellent effect (as distinctfrom insecticidal activity) was made.

In a later publication by Cavill, G. W. K., et al. (Tetrahedron38:1931-1938 (1982)), the presence of dihydronepetalactones in thedefensive secretion of an ant was again reported, but the authorsconcluded that the compound iridodial (and not a dihydronepetalactone)was the basic repellent constituent.

Most recently, Jefson, M., et al. (J. Chemical Ecology 9:159-180 (1983))described the repellent effect of dihydronepetalactone. Initialrepellency caused by the undiluted compound was measured with respect tothe ant species Monomorium destructor during feeding. After 25 secondsof exposure to the pure dihydronepetalactone, approximately 50-60% ofthe ants ceased to feed. However, further analyses of the repellencyover a longer time course were not presented, nor were analyses withanything other than the pure undiluted compound. Repellency observedover such short periods of time (seconds) with concentrated chemicals isinsufficient to allow prediction of efficacy in practical applicationssuch as in topical insect repellents.

There is consequently a continuing need for a biologically basedcompound having improved insect repellent properties (with respect toDEET) and which is substantially non-toxic or only mildly toxic tohumans. Preferred repellents will have activity against a wide varietyof insects, including biting insects, wood-boring insects, noxiousinsects, household pests, and the like. Applicants have found thatdihydronepetalactones perform well as a new class of effective insectrepellent compounds without the disadvantageous propertiescharacteristic of prior-art compositions.

SUMMARY OF THE INVENTION

One embodiment of this invention is an insect repellent composition orarticle that contains a dihydronepetalactone, or a mixture ofdihydronepetalactone stereoisomers, represented by the general formula:

Another embodiment of this invention is a process for fabricating aninsect repellent composition or an insect repellent article ofmanufacture by providing as the composition or article, or incorporatinginto the composition or article, a diihydronepetalactone, or a mixtureof dihydronepetalactone stereoisomers, as described above. A furtherembodiment of this invention is a method of imparting, augmenting orenhancing the insect repellent effect of an article by incorporatinginto the article a diihydronepetalactone, or a mixture ofdihydronepetalactone stereoisomers, as described above.

Yet another embodiment of this invention is the use of adiihydronepetalactone, or a mixture of dihydronepetalactonestereoisomers, as described above as an insect repellent, and thus in amethod of repelling insects, the insects are exposed to adiihydronepetalactone, or a mixture of dihydronepetalactonestereoisomers, as described above.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the chemical structures of the naturally-occurring iridoid(methylcyclopentanoid) nepetalactones.

FIG. 2 shows the total ion chromatograms from combined gaschromatography/mass spectrometry (GC-MS) analysis of a distillednepetalactone-enriched fraction from commercially-available catmint oil(A), together with that of the material produced from this fraction byhydrogenation (B).

FIG. 3 shows the mass spectra of the major constituents of thenepetalactone-enriched fraction (A) and the hydrogenated material (B)identified by GC-MS analysis.

FIG. 4 shows the ¹³C NMR analysis performed on a distillednepetalactone-enriched fraction of commercially-available catmint oil.

FIG. 5 shows the ¹³C NMR spectrum obtained from analysis of thedihydronepetalactones produced by hydrogenation of a distillednepetalactone-enriched fraction of commercially-available catmint oil.

FIG. 6 shows the distribution of landing density of female Aedesaegyptii mosquitoes on membranes treated with dihydronepetalactones overtime in an in vitro repellency test.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “nepetalactone” refers to the compound havingthe general structure:

Four chiral centers are present within the methylcyclopentanoid backboneof nepetalactone at carbons 4, 4a, 7 and 7a as shown above;(7S)-nepetalactones is produced by several plants and insects.Dihydronepetalactones are defined by Formula 1:

wherein 1, 5, 6 and 9 indicate the four chiral centers of the moleculeand the structure encompasses all possible stereoisomers ofdihydronepetalactone. The structures of dihydronepetalactonestereoisomers that may be derived from (7S)-nepetalactones are shownbelow.

As used herein, the term “dihydronepetalactones” or“dihydronepetalactone mixtures” refers to any mixture ofdihydronepetalactone stereoisomers. The molar or mass composition ofeach of these isomers relative to the whole dihydronepetalactonecomposition can be variable. Dihydronepetalactones are abbreviated as“DHN”.

As used herein, the term “insect” refers to any member of a large groupof invertebrate animals characterized, in the adult state (non-adultinsect states include larva and pupa) by division of the body into head,thorax, and abdomen, three pairs of legs, and, often (but not always)two pairs of membranous wings. This definition therefore includes avariety of biting insects (e.g., ants, bees, black flies, chiggers,fleas, green head flies, mosquitoes, stable flies, ticks, wasps),wood-boring insects (e.g., termites), noxious insects (e.g., houseflies,cockroaches, lice, roaches, wood lice), and household pests (e.g., flourand bean beetles, dust mites, moths, silverfish, weevils).

As used herein, the term “host” hereinafter refers to any plant oranimal affected by insects. Typically, hosts are considered to beinsect-acceptable food sources or insect-acceptable habitats.

As used herein, the term “insect susceptible article” will refer to anyitem of commerce created by man, which is affected by insects. This mayinclude buildings, furniture, and the like. Typically, these articles ofmanufacture are considered to be insect-acceptable food sources orinsect-acceptable habitats.

As used herein, the term “insect repellent” or “insect repellentcomposition” or “repellent composition” will refer to a compound orcomposition, which deters insects from their preferred hosts orinsect-suitable articles of manufacture. Most known repellents are notactive poisons at all, but rather prevent damage to plants/animals orarticles of manufacture by making insect food sources or livingconditions unattractive or offensive. Typically, insect repellents are acompound or composition that can be either topically applied to thehost; or, the compound or composition is incorporated into an insectsusceptible article to produce an insect repellent article that detersinsects from the nearby 3-dimensional space in which the host or articleexists. In either case, the affect of the insect repellent is to drivethe insects away from or to reject: 1.) the host, thereby minimizing thefrequency of insect “bites” to the host; or 2.) the insect susceptiblearticle, thereby protecting the article from insect damages. Repellentsmay be in the form of gases (olfactory), liquids, or solids (gustatory).

Some examples of well-known insect repellents include: benzil; benzylbenzoate; 2,3,4,5-bis(butyl-2-ene)tetrahydrofurfural (MGK Repellent 11);butoxypolypropylene glycol; N-butylacetanilide;normal-butyl-6,6-dimethyl-5,6-dihydro-1,4-pyrone-2-carboxylate(Indalone); dibutyl adipate; dibutyl phthalate; di-normal-butylsuccinate (Tabatrex); N,N-diethyl-meta-toluamide (DEET); dimethylcarbate (endo,endo)-dimethylbicyclo[2.2.1]hept-5-ene-2,3-dicarboxylate); dimethyl phthalate;2-ethyl-2-butyl-1,3-propanediol; 2-ethyl-1,3-hexanediol (Rutgers 612);di-normal-propyl isocinchomeronate (MGK Repellent 326);2-phenylcyclohexanol; p-methane-3,8-diol, and normal-propylN,N-diethylsuccinamate. Standard repellents for mosquitoes, ticks, andthe like are citronella oil (discussed below), dimethyl phthalate,normal-butylmesityl oxide oxalate and 2-ethyl hexanediol-1,3 (See,Kirk-Othmer Encyclopedia of Chemical Technology, 2^(nd) Ed., Vol. 11:724-728; and The Condensed Chemical Dictionary, 8^(th) Ed., p 756).

In addition to the chemical compositions above, a variety of effectiveinsect repellents consist of essential oils and/or active ingredients ofessential oils. “Essential oils” are defined as any class of volatileoils obtained from plants possessing the odor and other characteristicproperties of the plant. Examples of repellent compounds that areessential oils include: almond bitter oil, anise oil, basil oil, bayoil, caraway oil, cardamom oil, cedar oil, celery oil, chamomile oil,cinnamon oil, citronella oil, clove oil, coriander oil, cumin oil, dilloil, eucalyptus oil, fennel oil, ginger oil, grapefruit oil, lemon oil,lime oil, mint oil, parsley oil, peppermint oil, pepper oil, rose oil,spearmint oil (menthol), sweet orange oil, thyme oil, turmeric oil, andoil of wintergreen. Examples of active ingredients in essential oilsare: citronellal, methyl salicylate, ethyl salicylate, propylsalicylate, citronellol, safrole, and limonene.

In contrast to an “insect repellent”, an “insecticide” is a compound ormixture, which is capable of poisoning an insect via its oral ingestion,by contact with the insect cuticle, or by fumigant action through theair. Thus, an insecticide is a type of pesticide designed to controlinsect life, which is harmful to man (i.e., directly harmful as diseasevectors, or indirectly harmful by destruction of crops, food products,or textile fabrics). Several well-known insecticides include: inorganiccompounds (such as arsenic, lead and copper); naturally occurringorganic compounds (such as rotenone, pyrethrins, nicotine, coppernaphthenate and petroleum derivatives); and synthetic organic compounds(such as DDT, dieldrin, endrin, chlordane, lindane, para-dichlorobenzeneand parathion).

As used herein, the term “potentiated insect repellent composition”refers to a repellent composition, which produces a result substantiallyin excess of that which reasonably could be expected or predicted fromthe known effect of the components either alone or additively. In thepresent invention, a potentiated insect repellent composition willinclude dihydronepetalactones or a mixture thereof, and at least oneother insect repellent compound that is not itself dihydronepetalactone(sometimes referred to as a non-dihydronepetalactone insect repellentcompound).

An “insect repellent composition” can be used as a component of an“insect repellent article”, wherein the term “insect repellent article”refers to an article of manufacture possessing insect repellency that isenhanced, altered, or augmented by the insect repellent composition. Asused herein with respect to insect repellency, the terms “alter” and“modify” in their various forms refer to a means of supplying orimparting insect repellency to a composition, or augmenting the existinginsect repellency characteristics where natural repellency is deficientin some regard, or supplementing the existing insect repellency tomodify its quality, or character. The term “enhance” is intended to meanthe intensification (without effecting a change in kind or quality ofrepellency) of one or more repellency properties in an insect repellentcomposition or insect repellent article.

In a preferred embodiment, the insect repellent composition of thisinvention also functions as a fragrance composition since it is capableof imparting a pleasing fragrance or aroma to the insect repellentcomposition or to an insect repellent article. Dihydronepetalactones areuseful in an insect repellent composition or article to enhance, alter,or augment the overall aroma or fragrance of the composition or article.With respect to fragrance, the terms “alter” and “modify” in theirvarious forms refer to a means of supplying or imparting a fragrance oraroma character or note to otherwise bland substances or augmenting theexisting aroma characteristics where natural aroma is deficient in someregard or supplementing the existing aroma impression to modify itsquality, character, or aroma. The term “enhance” is intended to mean theintensification (without effecting a change in kind or quality of aroma)of one or more aroma nuances and their organoleptic impression of afragrance, perfume composition, or one or more perfumed articles.

The term “fragrance composition” is used herein to mean a mixture oforganic compounds including, for example, alcohols, aldehydes, ketones,nitrites, esters, lactones, natural essential oils, synthetic essentialoils, and mercaptans, which are admixed so that the combined odors ofthe individual components produce a pleasant or desired fragrance. Suchcompositions usually contain: (1) the main note or the “bouquet” orfoundation stone of the composition; (2) modifiers which round off andaccompany the main note; (3) fixatives which include odorous substanceswhich lend a particular note to the perfume throughout all stages ofevaporation and substances which retard evaporation; and (4) top noteswhich are usually low-boiling, fresh-smelling materials.

In fragrance or aroma compositions, the individual component willcontribute its particular olfactory characteristics, but the overalleffect of the composition will be the sum of each of the effects of eachof the ingredients. Thus, the dihydronepetalactones of this invention ormixtures thereof can be used to alter the aroma characteristics of suchcompositions, for example, by highlighting or moderating the olfactoryreaction contributed by another ingredient in the composition.

Dihydronepetalactones are known in the literature, for example as minorconstituents of the essential oils of several labiate plants of thegenus Nepeta (Regnier, F. E., et al. Phytochemistry6:1281-1289 (1967);DePooter, H. L., et al. Flavour and Fragrance Journal 3:155-159 (1988);Handjieva, N. V. and S. S. Popov J. Essential Oil Res. 8:639-643(1996)). Additionally, dihydronepetalactones have been identified asconstituents of the defensive secretions of certain insects, includingrove beetles (Jefson, M., et al. J. Chem. Ecol. 9:159-180 (1983)) andants, specifically Iridomyrmex species (Cavill, G. W. K. and D. V.Clark. J. Insect Physiol. 13:131-135 (1967)). In those species thatpossess dihydronepetalactones, it has been proposed that they arebiosynthetically derived from the iridoid monoterpene iridodial.

The chemical synthesis of dihydronepetalactones and their relatediridoid monoterpenoid compounds has been described and found to beconducted in a variety of ways. The following are useful referencesrelating to synthesis:

-   -   1) Abelman, M. M. et al. J. Am. Chem. Soc. 104(14):4030-2 (1982)    -   2) Fleming, I. and N. K. Terrett. Tetrahedron Lett. 25(44):        5103-5104 (1984); J. Chem. Soc., Perkin Trans. 1:2645-2650        (1998).    -   3) Lee, E. and C. H. Yoon. J. Chem. Soc., Chem. Commun. 4:        479-81 (1994).    -   4) Nagata, H. and K. Ogasawara. Tetrahedron Lett. 40(36):        6617-6620 (1999).    -   5) Nangia, A. et al. Tetrahedron Lett. 35(22): 3755-8 (1994).    -   6) Tanimori, S. and M. Nakayama. Agric. Biol. Chem. 55(4):        1181-1184 (1991).    -   7) Uyehara, T. et al. J. Chem. Soc., Chem. Commun. 2:113-14        (1989); Tennen Yuki Kagobutsu Toronkai Koen Yoshishu 32:

441-6 (1990); J. Org. Chem. 57(11): 3139-3145 (1992).

-   -   8) Wolinsky, J. and E. J. Eustace. J. Org. Chem. 37(21): 3376-8        (1972).    -   9) Wolinsky, J. and D. L. Nelson. Tetrahedron 25(17): 3767-74        (1969).

One preferred and convenient method for synthesis of thedihydronepetalactone mixtures of the present invention is byhydrogenation of nepetalactone. Catalysts such as platinum oxide andpalladium supported on strontium carbonate give dihydronepalactone in24-90% yields (Regnier, F. E., et al. Phytochemistty 6:1281-1289(1967)). Nepetalactone is a known material that can be convenientlyobtained in relatively pure form from the essential oils isolated byvarious means from plants of the genus Nepeta (catmints). Isolation ofsuch oils is well known in the art, and examples of methodology for oilextraction include (but are not limited to) steam distillation, organicsolvent extraction, microwave-assisted organic solvent extraction,supercritical fluid extraction, mechanical extraction and enfleurage(initial cold extraction into fats followed by organic solventextraction).

The essential oils isolated from different Nepeta species are well knownto possess different proportions of each naturally-occurringstereoisomer of nepetalactone (Regnier, F. E., et al. Phytochemistry6:1281-1289 (1967); DePooter, H. L., et al. Flavour and FragranceJournal 3:155-159 (1988); Handjieva, N. V. and S. S. Popov. J. EssentialOil Res. 8:639-643 (1996)). Thus, from oil derived from any Nepetaspecies containing a mixture of nepetalactones, a mixture ofdihydronepetalactone stereoisomers will be generated upon hydrogenation.Four chiral centers are present within the methylcyclopentanoid backboneof the nepetalactone at carbons 4, 4a, 7 and 7a as shown:

Thus it is clear that a total of eight pairs of dihydronepetalactoneenantiomers are possible after hydrogenation. Of these, the naturallyoccurring stereoisomers described thus far are(9S)-dihydronepetalactones. Preferred repellent materials in accordancewith the present invention include a mixture of any or all of thepossible stereoisomers of dihydronepetalactone. More preferred repellentmaterials include a mixture of (9S)-dihydronepetalactones. Mostpreferred are (9S)-dihydronepetalactone stereoisomers derived from(7S)-nepetalactones. This includes the compounds commonly known ascis,trans-nepetalactone, cis,cis-nepetalactone, trans,cis-nepetalactone,and trans,trans-nepetalactone, as illustrated in FIG. 1.

Upon completion of the hydrogenation reaction, the resulting mixture ofisomer products may be separated by a conventional method (e.g.,preparative liquid chromatography) to yield each highly purified pair ofdihydronepetalactone diastereomers.

In addition to variation in nepetalactone stereoisomer content betweendifferent Nepeta species, intra-species variation is also known toexist. Plants of a given species may produce oils with differentcompositions depending on the conditions of their growth or growth stageat harvest. Additionally, within a single species, Nepeta racemosa,variation in oil composition independent of growth conditions or growthstage at harvest has been demonstrated (Clark, L. J., et al. The PlantJournal, 11:1387-1393 (1997)). Plants of a single species exhibitingdifferent oil compositions are termed chemo types, and it has been shownthat in Nepeta racemosa, chemo types exhibiting marked differences inthe proportion of different nepetalactone stereoisomers exist (Clark, L.J.,et al., supra). Thus, the preferred process for producing specificdihydronepetalactone enantiomers would be hydrogenation of an oil from aNepeta chemo type known to contain specific nepetalactone stereoisomers.

The preferred process for producing the dihydronepetalactonesrepresented by Formula I in the present invention, therefore, is byhydrogenation of nepetalactones from plants with oils of definednepetalactone stereoisomer content, an industrially advantageousapproach in terms of production cost and its biological basis. Otherprocesses are as disclosed in U.S. Provisional Application No.60/369,470, filed Apr. 3, 2002.

The dihydronepetalactones of the present invention possess uniqueproperties of insect repellency and are particularly effective against awide spectra of common insect pests, including biting insects,wood-boring insects, noxious insects, and household-pests.

The insect repellent compositions of this invention containingdihydronepetalactones or mixtures thereof are effective against avariety of insects, which interfere with human society. These insectsinclude a variety of biting insects (e.g., ants, bees, black flies,chiggers, fleas, green head flies, mosquitoes, stable flies, ticks,wasps), wood-boring insects (e.g., termites), noxious insects (e.g.,houseflies, cockroaches, lice, roaches, wood lice), and household pests(e.g., flour and bean beetles, dust mites, moths, silverfish, weevils).In the case of mosquitoes, which convey pathogenic microbes, theserepellent properties are additionally effective for preventing infectionwith such diseases.

A wide variety of compounds possess insect repellent and/or mosquitorepellent activity, as evidenced by: 1.) the diversity of chemicalstructures reported by the USDA to contain repellent activity (ChemicalsEvaluated as Insecticides and Repellents at Orlando, Fla., compiled byW. V. King, U.S. Department of Agriculture, Agricultural ResearchService, Agriculture Handbook No. 69); and 2.) by the variety of insectrepellant active materials present in insect repellent formations (See,e.g., European patent applications 97,812 and 97,813, and U.S. Pat. No.4,127,672, U.S. Pat. No. 4,756,905, U.S. Pat. No. 5,465,685, U.S. Pat.No. 5,489,433, U.S. Pat. No. 5,565,208, U.S. Pat. No. 5,672,337 and U.S.Pat. No. 5,716,602). In general, activity is found in alcohols, amides,esters, ketones, acids, lactones, and lactams; and to some degree,repellency activity does appear to depend on the physical properties ofthese compounds.

One property that is important to overall insect repellency is surfaceactivity, as most (if not all) repellents contain both polar andnon-polar regions in their structure. A second property is volatility.Insect repellents form an unusual class of compounds where evaporationof the active ingredient from the host's skin surface or from the insectrepellent article is necessary for effectiveness, as measured by thehost's protection from insect bites or the article's protection frominsect damage. In the case of a topical insect repellent, a certainminimum concentration of repellent is needed in the air space directlyabove the skin surface of the host in order to repel insects, and thisconcentration is a measure of the potency of the repellent. However,evaporation rate is also affected by the rate of skin absorption—in mostcases, penetration into and through the skin is an undesirable mode ofloss of compound from the skin surface. Similar considerations must bemade for insect repellent articles, concerning the minimum concentrationof repellent required in the three-dimensional air space surrounding thearticle itself.

A variety of strategies are available to researchers attempting tobalance these properties of evaporation (and optionally, penetration).First, it is possible to find a single active ingredient having theright balance of physical properties. Alternatively, the activeingredient could be formulated with polymers and inert ingredients addedto the active ingredient for the purpose of modifying the persistence ofthe active ingredient on the host's skin surface or within the insectrepellent article. However, adding inert ingredients to the activeingredient limits the number of molecules of active ingredient on thesurface of the repellent film or article. Since a molecule must be onthe surface in order to evaporate, the evaporation rate is lowered. Thiscarries with it the negative consequence of diluting the concentrationof active ingredient that can be applied to the host's skin surface orthat is present on the surface of an insect repellent article. This, inturn, reduces the overall potency of a formulation containing inertingredients. In a third alternative, the active ingredient can becontained in microcapsules to control rates of loss from the host's skinsurface or insect repellent article. Finally, another technique oflimiting the evaporation rate of active ingredient is to synthesize aprecursor molecule, which slowly disintegrates on the skin surface orinsect repellent article to release the active ingredient.

For example, release of the active ingredient may be, for example, bysub-micron encapsulation, in which the active ingredient is encapsulated(surrounded) within a skin nourishing protein just the way air iscaptured within a balloon. The protein may be used at, for example, a20% concentration. An application of repellent contains many of theseprotein capsules that are suspended in either a water-based lotion, orwater for spray application. After contact with skin the proteincapsules begin to breakdown releasing the encapsulateddihydronepetalactone. The process continues as each microscopic capsuleis depleted then replaced in succession by a new capsule that contactsthe skin and releases its active ingredient. The process may take up to24 hours for one application. Because protein's adherence to the skin isso effective, these formulas are very resistant to perspiration(sweat-off), and water. When applied they are dry and comfortable withno greasiness. This system results in very effective protection, but itis only effective when used on skin because clothing does not have thecapability to release the proteins. An alternative system uses a polymerto encase the repellent, which slows down early evaporation leaving moredihydronepetalactone available for later evaporation. This system canoften increase a repellent's length of effectiveness by 25% to 50% overcomparable non-entrapped products, but often feels greasy because of thepresence of the polymer. In another alternative, a synergist is used tokeep stimulating the evaporation of the dihydronepetalactone in thecomposition.

Regardless of the particular strategy applied to control volatility ofan insect repellent, each repellent must have a minimum effectiveevaporation rate (MEER) from the skin surface or insect repellentarticle to maintain the necessary minimum concentration of repellent inthe air space directly above the skin surface/article for effectiveinsect repellency. An evaporation rate greater than the minimumeffective evaporation rate (MEER) results in a significant andundesirable mode of loss. The issue is further complicated, however,since the MEER will change as a function of conditions in the field.Both the avidity and biting tendency of an insect and the concentrationof insects in the host's environment must be considered. For example, asthe avidity of mosquitoes increases, a higher MEER will be required. Inan environment having a low concentration of mosquitoes where thosemosquitoes are not hungry, the MEER could be as low as 2, or morecommonly 5, or 6. In contrast, in an environment having a highconcentration of hungry mosquitoes, the MEER might be as high as 12-15.Preferred in the present invention are insect compositions wherein theskin surface evaporation rate is at least equal to a minimum effectiveevaporation rate over a period of time where a preferred period of timeis about 5 hours.

In the present invention, a variety of carriers or diluents for theabove-disclosed dihydronepetalactones can be used. The carrier allowsthe formulation to be adjusted to an effective concentration ofrepellant molecules. When formulating a topical insect repellent,preferably, the repellant molecules are mixed in a dermatologicallyacceptable carrier. The carrier may further provide water repellency,prevent skin irritation, and/or soothe and condition skin. Factors toconsider when selecting a carrier(s) for any formulation of insectrepellent include commercial availability, cost, repellency, evaporationrate, odor, and stability. Some carriers can themselves have repellentproperties.

For the present invention, the specific choice of a carrier, if any, tobe utilized in achieving the desired intimate admixture with the finalproduct can be any carrier conventionally used in insect repellentformulations. The carrier, moreover, should preferably also be one thatwill not be harmful to the environment. Accordingly, the carrier can beany one of a variety of commercially available organic and inorganicliquid, solid, or semi-solid carriers or carrier formulations usable informulating insect repellent products. For example the carrier mayinclude silicone, petrolatum, lanolin or many of several otherwell-known carrier components.

Examples of organic liquid carriers include liquid aliphatichydrocarbons (e.g., pentane, hexane, heptane, nonane, decane and theiranalogs) and liquid aromatic hydrocarbons. Examples of other liquidhydrocarbons include oils produced by the distillation of coal and thedistillation of various types and grades of petrochemical stocks,including kerosene oils, which are obtained by fractional distillationof petroleum. Other petroleum oils include those generally referred toas agricultural spray oils (e.g., the so-called light and medium sprayoils, consisting of middle fractions in the distillation of petroleumand which are only slightly volatile). Such oils are usually highlyrefined and may contain only minute amounts of unsaturated compounds.Such oils, moreover, are generally paraffin oils and accordingly can beemulsified with water and an emulsifier, diluted to lowerconcentrations, and used as sprays. Tall oils, obtained from sulfatedigestion of wood pulp, like the paraffin oils, can similarly be used.Other organic liquid carriers can include liquid terpene hydrocarbonsand terpene alcohols such as alpha-pinene, dipentene, terpineol, and thelike.

Other carriers include silicone, petrolatum, lanolin, liquidhydrocarbons, agricultural spray oils, paraffin oil, tall oils, liquidterpene hydrocarbons and terpene alcohols, aliphatic and aromaticalcohols, esters, aldehydes, ketones, mineral oil, higher alcohols,finely divided organic and inorganic solid materials.

In addition to the above-mentioned liquid hydrocarbons, the carrier cancontain conventional emulsifying agents which can be used for causingthe dihydronepetalactone compounds to be dispersed in, and diluted with,water for end-use application.

Still other liquid carriers can include organic solvents such asaliphatic and aromatic alcohols, esters, aldehydes, and ketones.Aliphatic monohydric alcohols include methyl, ethyl, normal-propyl,isopropyl, normal-butyl, sec-butyl, and tert-butyl alcohols. Suitablealcohols include glycols (such as ethylene and propylene glycol) andpinacols. Suitable polyhydroxy alcohols include glycerol, arabitol,erythritol, sorbitol, and the like. Finally, suitable cyclic alcoholsinclude cyclopentyl and cyclohexyl alcohols.

Conventional aromatic and aliphatic esters, aldehydes and ketones can beused as carriers, and occasionally are used in combination with theabove-mentioned alcohols. Still other liquid carriers include relativelyhigh-boiling petroleum products such as mineral oil and higher alcohols(such as cetyl alcohol). Additionally, conventional or so-called“stabilizers” (e.g., tert-butyl sulfinyl dimethyl dithiocarbonate) canbe used in conjunction with, or as a component of, the carrier orcarriers comprising the compositions of the present invention.

Solid carriers, which can be used in the compositions of the presentinvention include finely divided organic and inorganic solid materials.Suitable finely divided solid inorganic carriers include siliceousminerals such as synthetic and natural clay, bentonite, attapulgite,fuller's earth, diatomaceous earth, kaolin, mica, talc, finely dividedquartz, and the like, as well as synthetically prepared siliceousmaterials, such as silica aerogels and precipitated and fume silicas.Examples of finely divided solid organic materials include cellulose,sawdust, synthetic organic polymers, and the like. Examples ofsemi-solid or colloidal carriers include waxy solids, gels (such aspetroleum jelly), lanolin, and the like, and mixtures of well-knownliquid and solid substances which can provide semi-solid carrierproducts, for providing effective repellency within the scope of theinstant invention.

Insect repellent compositions of the present invention containing thedihydronepetalactones may also contain adjuvants known in the art ofpersonal care product formulations, such as thickeners, bufferingagents, chelating agents, preservatives, fragrances, antioxidants,gelling agents, stabilizers, surfactants, emolients, coloring agents,aloe vera, waxes, other penetration enhancers and mixtures thereof, andtherapeutically or cosmetically active agents.

Additionally, the compositions of the present invention may also containother adjuvants such as one or more therapeutically or cosmeticallyactive ingredients. Exemplary therapeutic or cosmetically activeingredients useful in the compositions of the invention includefungicides, sunscreening agents, sunblocking agents, vitamins, tanningagents, plant extracts, anti-inflammatory agents, anti-oxidants, radicalscavenging agents, retinoids, alpha-hydroxy acids, emollients,antiseptics, antibiotics, antibacterial agents or antihistamines, andmay be present in an amount effective for achieving the therapeutic orcosmetic result desired.

Dihydronepetalactones may be utilized in the present inventionindividually or combined in any proportion. As is conventional in theart, the desired amount of an insect repellent composition to be addedto a given insect susceptible article with properties of insectrepellency is determined by the nature of the product and other factors.These factors include both considerations of cost and the nature of theother ingredients in the insect repellent composition or repellentarticle, their amounts, and the desired repellency effect. In general,the composition of the repellent should contain sufficient amounts ofactive insect repellant material to be efficacious in repelling theinsect from the host over a prolonged period of time (preferably, for aperiod of at least several hours).

The amount of each dihydronepetalactone of Formula I or mixtures thereofin an insect repellent composition or repellent article in accordancewith the present invention will generally not exceed about 80% by weightbased on the weight of the final product, however, greater amounts maybe utilized in certain applications and this amount is not limiting.More preferably, a suitable amount of dihydronepetalactone will be atleast about 0.001% by weight and preferably about 0.01% up to about 50%by weight; and more preferably, from about 0.01% to about 20% weightpercent, based on the weight of the composition or article. Specificcompositions will depend on the intended use.

The dihydronepetalactone repellent compositions of the present inventioncan be formulated without a carrier and be effective. More often,however, the insect repellent composition will include a carrier andcontain about 0.001-50% weight of the disclosed compounds, and suchcompound is usually in intimate mixture with the carrier to bring theactive material into position for repelling common insect pests, such asbiting insects, wood-boring insects, noxious insects, household pests,and the like.

The compositions of the invention may be formulated and packaged so asto deliver the product in a variety of forms including as a solution,suspension, cream, ointment, gel, film or spray, depending on thepreferred method of use. The carrier may be an aerosol compositionadapted to disperse the dihydronepetalactone into the atmosphere bymeans of a compressed gas.

In some cases, the dihydronepetalactone repellent compositions of thepresent invention can be formulated with at least one other insectrepellent that is not itself dihydronepetalactone to create apotentiated insect repellent composition (see, for example, U.S. Pat.No. 4,869,896). In this case, the effect of the dihydronepetalactone andthe at least one other insect repellent produce a repellency resultsubstantially in excess of that which reasonably could be expected orpredicted from the known effect of the components either alone oradditively. Exemplary other insect repellent compounds which may be usedin a potentiated insect repellent composition with dihydronepetalactonesinclude, but are not limited to: benzil; benzyl benzoate;2,3,4,5-bis(butyl-2-ene)tetrahydrofurfural (MGK Repellent 11);butoxypolypropylene glycol; N-butylacetanilide;normal-butyl-6,6-dimethyl-5,6-dihydro-1,4-pyrone-2-carboxylate(Indalone); dibutyl adipate; dibutyl phthalate; di-normal-butylsuccinate (Tabatrex); N,N-diethyl-meta-toluamide (DEET); dimethylcarbate (endo,endo)-dimethylbicyclo[2.2.1]hept-5-ene-2,3-dicarboxylate); dimethyl phthalate;2-ethyl-2-butyl-1,3-propanediol; 2-ethyl-1,3-hexanediol (Rutgers 612);di-normal-propyl isocinchomeronate (MGK Repellent 326);2-phenylcyclohexanol;normal-propyl N,N-diethylsuccinamate,1-piperidinecarboxylic acid 2-(2-hydroxymethyl) 1-methylpropylester(Bayrepel) and p-menthane-3,8-diol. Preferred is a potentiated insectrepellent composition comprising dihydronepetalactones (or a mixturethereof) and DEET, Bayrepel or p-menthane-3,8-diol. Most preferred is apotentiated insect repellent composition comprisingdihydronepetalactones (or a mixture thereof) and other natural repellentmolecules such as p-menthane-3,8-diol.

Desirable properties of a topical insect repellent article include lowtoxicity, resistance to loss by water immersion or sweating, low or noodor or at least a pleasant odor, ease of application, and rapidformation of a dry tack-free surface film on the host's skin. In orderto obtain these properties, the formulation for a topical insectrepellent article should permit insect-infested animals (e.g., dogs withfleas, poultry with lice, cows with ticks, and humans) to be treatedwith an insect repellent composition of the present invention bycontacting the skin, fur or feathers of such an animal with an effectiveamount of the repellent article for repelling the insect from the animalhost. Thus, dispersing the article into the air or dispersing thecomposition as a liquid mist or fine dust will permit the repellentcomposition to fall on the desired host surfaces. Likewise, directlyspreading of the liquid/semi-solid/solid repellent article on the hostis an effective method of contacting the surface of the host with aneffective amount of the repellent composition.

Embodiments of the present invention which may be used as a topicalinsect repellent articles, include (but are not limited to): colognes,lotions, sprays, creams, gels, ointments, bath and shower gels, foamproducts (e.g., shaving foams), makeup, deodorants, shampoo, hairlacquers/hair rinses, and personal soap compositions (e.g., hand soapsand bath/shower soaps).

This invention also relates to the use of dihydronepetalactone mixturesas insect repellents in a variety of articles which are susceptible toattack by insects. These outdoor consumable products will generally, butnot necessarily, comprise an insect repellent composition of theinvention, but will contain an effective amount of dihydronepetalactone.Typical articles that can be improved by the use ofdihydronepetalactones and mixtures thereof include, but are not limitedto: air fresheners, candles, other scented articles, fibers, sheets,cloth [e.g., for clothing, nettings (mosquito netting), and otherfabrics], paper, paint, ink, clay, woods, furniture (e.g., for patiosand decks), carpets, sanitary goods, plastics, polymers, and the like.

The dihydronepetalactone compositions of this invention may be blendedwith polymers, which may also involve a controlled release systems.Compatible polymers may or may not be biodegradable. Exemplary polymersare high density polyethylene or low density polyethylene, biodegradablethermoplastic polyurethanes, biodegradable ethylene polymers, andpoly(epsilon caprolactone) homopolymers and compositions containing thesame as disclosed in U.S. Pat. No. 4,496,467, U.S. Pat. No. 4,469,613and U.S. Pat. No. 4,548,764.

Dihydronepetalactones are particularly advantageous for use as repellentmaterials in preparations of the present invention for a variety ofreasons.

First, the compounds are cost effective to produce, an importantconsumer consideration when choosing an effective repellent. Manycommerically available repellent products are only effective inrelatively concentrated form, including as much as 5-30% (or more)repellent in a carrier, based on total weight. U.S. Pat. No. 4,416, 881to McGovern et al., for example, discloses repellent concentrations of6.25-25% repellent in a carrier.

Secondly, the compounds are known natural compounds, thus overcomingconcerns raised against synthetic chemicals such as DEET as the primaryactive ingredient in repellent compositions.

Finally, in addition to the natural repellent properties of thedihydronepetalactone mixtures thus prepared, the compositions alsopossess a unique and pleasant fragrance. The fragrance notes of thesubject compounds make them useful in imparting, altering, augmenting orenhancing the overall olfactory component of an insect repellentcomposition or article, for example, by utilizing or moderating theolfactory reaction contributed by one or more other ingredients in thecomposition. Specifically, the dihydronepetalactones of the invention ormixtures thereof may be utilized to either mask or modify the odorcontributed by other ingredients in the formulation of the finalrepellent composition or article, and/or to enhance consumer appeal of aproduct by imparting a characteristic perfume or aroma. It is expectedthat the pleasant fragrance of dihydronepetalactones will possess muchgreater appeal to consumers than other insect repellent compounds,particularly DEET.

Dihydronepetalactones and their uses are also described in U.S. Ser. No.10/349,865, filed Jan. 23, 2003, which is incorporated in its entiretyas a part hereof for all purposes.

EXAMPLES

The present invention is further defined in the following examples. Itshould be understood that these examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. The notation below of w/v refers to the weight of the activeingredient in grams in 100 mL of solution.

The meaning of abbreviations is as follows: “h” means hour(s), “min”means minute(s), “sec” means second(s), “d” means day(s), “mL” meansmilliliters, “L” means liters, “m/z” means mass (m) to charge (z) ratio,“ppm” means parts per million, “mol %” means percentage expressed on amolar basis, “Hz” means Hertz (1/sec), and “psig” means pounds persquare inch guage.

Example 1 Preparation of Nepetalactones by Fractional Steam Distillationof Oil of Nepeta cataria

A sample of commercially-available catnip oil, prepared by steamdistillation of herbaceous material from the catmint Nepeta cataria, wasobtained (Berjé, Bloomfield, N.J., USA). Combined gaschromatography—mass spectrometry (GC-MS) of this oil indicated that theprincipal constituents were nepetalactone stereoisomers (data notshown).

The nepetalactone fraction was further purified by fractionaldistillation of this oil. FIG. 2A presents the GC-MS total ionchromatogram of the nepetalactone-enriched fraction prepared byfractional distillation of the commercial sample of Nepeta catariaessential oil. The conditions employed were: column HP5-MS, 25 m×0.2 mm;oven 120° C., 2 min, 15° C./min, 210° C., 5 min.; He @ 1 ml/min. Peakswith m/z 166 are nepetalactones). The unlabelled peaks correspond tominor sesquiterpenoid contaminants. In FIG. 3A, the mass spectrum of themajor peak (6.03 min, nepetalactone) in FIG. 2A is shown. ¹H and ¹³C NMRanalysis of the oil and the purified material was also carried out, andthe ¹³C data is presented (FIG. 4). The ¹³C chemical shifts for the fourpossible stereoisomers reported in the literature were compared to thespectra taken for the sample. Three stereoisomers were detected and theamounts were quantified based on the carbonyl region at around 170 ppm.The chemical shifts, for both the original oil and the enrichedmaterial, are provided in Table 1. Each carbon atom of nepetalatone isidentified, as shown in FIG. 4. TABLE 1 ¹³C Chemical Shifts and Mol %Values of Nepetalactone Stereoisomers Present in Commercial Sample ofEssential Oil of Catmint (Nepeta cataria) and in Fraction Purified bySteam Distillation PURIFIED FRACTION ESSENTIAL OIL trans, cis, trans-trans, cis- cis, cis- cis, trans- cis- cis, cis- ATOM δ (ppm) δ (ppm) δ(ppm) δ (ppm) δ (ppm) δ (ppm) a 170.9 170.1 170.8 170.1 b 133.7 135.9134.2 133.7 135.9 134.2 c 115.3 120.4 115.3 120.4 d 40.8 37.3 39.6 40.837.4 39.5 e 49.4 49.1 46.4 49.5 49.1 46.3 f 39.7 32.1 38.4 39.8 32.138.4 g 33.0 30.0 32.7 33.1 30.0 32.7 h 30.9 26.1 30.4 31.0 26.1 30.5 j20.2 17.5 17.1 20.3 17.6 17.2 i 15.4 14.2 14.7 15.5 14.2 14.8 Mol %80.20% 17.70% 2.10% 84.50% 14.30% 1.20%

This analysis indicated that in the oil, nepetalactones were present inthe following proportions: 80.2 mol % cis,trans-nepetalactone, 17.7 mol% trans,cis-nepetalactone and 2.1 mol % cis,cis-nepetalactone. The dataindicated the proportions of nepetalactones in the purified materialwere 84.5 mol % cis,trans-nepetalactone, 14.3 mol %trans,cis-nepetalactone and 1.2 mol % cis,cis-nepetalactone. GC-MSanalysis of this purified fraction indicated that it consistedpredominantly of these nepetalactones (m/z 166), accompanied by traceamounts of the sesquiterpenoids caryophyllene and humulene (data notshown).

Example 2 Preparation of Dihydronepetalactones

107 g of the distilled nepetalactone fraction of the catmint oilprepared as described in Example 1 was dissolved in ethanol (200 ml) andplaced in a Fisher-Porter bottle with 12.7 g 2% Pd/SrCO₃ (Aldrich41,461-1). The tube was evacuated and backfilled with H₂ two times, thencharged with H₂ at 30 psig. After 48 hr stirring at room temperature,the tube was vented and the contents filtered over Celite to removecatalyst. The solvent was removed under vacuum, yielding a clear oil.

GC-MS analysis (column HP5-MS, 25 m×0.2 mm; Oven 120° C., 2 min, 15°C./min, 210° C., 5 min.; He @ 1 ml/min) was conducted on this material.The total ion chromatogram is presented in FIG. 2B. This analysisindicated that the principal component (65.43% area; Rt 7.08 min)represented a dihydronepetalactone isomer, with m/z 168; the massspectrum of this component is presented in FIG. 3B. This spectrumcontains an ion with m/z 113, diagnostic for dihydronepetalactones(Jefson, M., et al. J. Chemical Ecology 9:159-180 (1983)). Fiveadditional peaks, representing the remaining dihydronepetalactonediastereomers which, might be derived from the three nepetalactonespresent in the starting material were also represented in thechromatogram. These occurred at Rt 5.41 min, 6.8% area, m/z 168; Rt 5.93min, area 1.2%, m/z 168; Rt 6.52 min, 4.88% area, mass 168; Rt 6.76 min,13.8% area, m/z 168 and Rt 7.13 min, 1.25% area, m/z 168. No residualnepetalactones were detected by GC-MS.

¹H, ¹³C and a series of 2D NMR analyses were also performed. Thecarbonyl region of the ¹³C NMR spectrum (FIG. 5) showed at least fivespin systems, one of them in larger amounts than the other four (ca.75%). Very little residual nepetalactone was detected.

Based on the analysis of coupling constants and the intensities of thedifferent NOE cross peaks observed, the stereochemistry of the principalcomponent of the material was determined to be the dihydronepetalactoneof Formula 2 (9S,5S,1R,6R)-5,9-dimethyl-3-oxabicyclo[4.3.0]nonan-2-one).

The distance between the methyl group (i) and proton (d) is longer thanthe distance between the methyl group (j) and the proton (e), anobservation consistent with the cis-trans stereochemical configuration.

The stereoisomer isodihydronepetalactone (9S,5R, 1R,6R)-5,9-dimethyl-3-oxabicyclo[4.3.0]nonan-2-one; (Formula 3) wassimilarly identified by ¹³C chemical shifts and is present in 3.6%.

Thus the GC-MS and NMR data indicate that hydrogenation of the mixtureof nepetalactone stereoisomers yielded the correspondingdihydronepetalactone diastereomers, as expected. The pair ofdiastereomers (Formula 2 and Formula 3) derived fromcis,trans-nepetalactone (84.5 Mol % of the starting material) were thepredominant dihydronepetalactones, at 78.6% of the mixture followinghydrogenation.

Example 3 Repellency Testing of a Dihydronepetalactone Mixture

The dihydronepetalactone mixture prepared in accordance with Example 2was evaluated for its repellent effects against female Aedes aegyptimosquitoes. These tests were carried out under contract by InsectControl & Research, Inc. (Baltimore, Md.).

Approximately 250 female Aedes aegypti mosquitoes were introduced into achamber containing 5 wells, each covered by a Baudruche membrane. Wellswere filled with bovine blood, containing sodium citrate (to preventclotting) and ATP (72 mg ATP disodium salt per 26 ml of blood), andheated to 37° C. A volume of 25 μl of isopropyl alcohol, containingputative repellent chemicals (Table 2), was applied to each membrane.TABLE 2 Experimental Design Applied for Repellency Testing PurposeCompound Concentration Untreated Control Isopropyl alcohol 100% PositiveControl Isopropyl alcohol with DEET 1.0% (w/v) Experimental SamplesIsopropyl alcohol with 1.0% (w/v) Dihydronepetalactone 2.5% (w/v) 5.0%(w/v)

After 5 min, 4 day-old female mosquitoes were added to the chamber. Thenumber of mosquitoes probing the membranes for each treatment wasrecorded at 2 min intervals over 20 min. All data presented is fromthree replicate experiments.

Table 3 presents the effect of dihydronepetalactone concentration withrespect to the amount of time taken before the female A. aegyptimosquitoes first probed each membrane. TABLE 3 Effect ofDihydronepetalactone Concentration on Mean Time to “First Probing”Repellent Concentration Mean Time (min) Isopropyl alcohol (untreated 4.6control)   1% DEET (positive control) 12   1% DHN 8 2.5% DHN 9.3   5%DHN 18

Mosquitoes began landing on the untreated control well within 4.6 min.Dihydronepetalactone at 5% concentration was found to discouragemosquito “first probing” for approximately 18 min, compared to 12 minfor DEET (at 1% w/v). Lower concentrations of dihydronepetalactone (1%and 2.5% w/v) were found to inhibit first probing for an average of 8and 9.3 min, respectively.

The distribution of landing density by female A. aegypti on membranestreated with dihydronepetalactones was analyzed over time, as showngraphically in FIG. 6. Dihydronepetalactone at 5% concentration wasfound to almost eliminate mosquito landings for 20 minutes, while DEET(1% w/v) permitted 2 mosquitoes to land in this experiment. Again, lowerconcentrations of dihydronepetalactone (1% and 2.5% w/v) were found toexhibit repellency (as compared to the untreated control), but at lowerlevels than the positive control (DEET at 1% w/v).

The total number of landings permitted on each membrane during thecourse of the experiments were determined, and the results aresummarized in Table 4. TABLE 4 Number of Landings Permitted According toRepellency Concentration Repellent Concentration Mean Number of LandingsIsopropyl alcohol (untreated 58.99 control)   1% DEET (positive control)4.66   1% DHN 14 2.5% DHN 6.33   5% DHN 0.33

Again, the data shows that at all concentrations testeddihydronepetalactone was repellent, although significantly increasedrepellency with respect to 1% DEET was observed only at 5% (w/v).

Cumulatively, this data show that at all three concentrations tested,treatment with dihydronepetalactone significantly reduced probing of themembranes over the course of the experiment (20 min) as compared to theisopropyl alcohol treatment. At concentrations of 2.5% and 5.0%,dihydronepetalactones had effectiveness as a repellent of mosquitoes forthe entire observation period. There was also a direct relationshipbetween dihydronepetalactone concentration and the time elapsed for thefirst mosquito to land and begin probing the membrane. Overall, the dataindicates that the mixture of dihydronepetalactones employed in theseexperiments was an effective repellent, although repellency equivalentto DEET was observed only with higher concentrations.

Example 4 Preparation of Dihydronepetalactones fromTrans,cis-nepetalactone

It has been shown that trans,cis-nepetalactone (4aS, 7S, 7aS- orE,Z-nepetalactone), is more effective than either the cis,trans-(Z,E-)nepetalactone or unfractionated catmint essential oil in repelling thegerman cockroach (Peterson, C. J. et al. (2002) Household and StructuralInsects, 95 (2), 377-380). Accordingly, we determined to purifytrans,cis-nepetalactone for hydrogenation to the correspondingdihydronepetalactones and repellency testing of these derivatives. Anumber of plants were grown from seed of the catmint Nepeta racemosa(Chiltern Seeds, Cumbria, UK). Leaf pairs plucked from individual plantswere immersed in ethyl acetate and after 2 h the solvent was removed andthe extracts analyzed by gas chromatography. Plants producingpreponderantly trans,cis-nepetalactone in their oils were thusidentified (Clark, L. J., et al. (1997) The Plant Journal,11:1387-1393), and grown to maturity. Leaf material from these plantswas harvested, freeze-dried, extracted into ethyl acetate, and theextracts concentrated. Nepetalactone was purified from the concentratedextract by silica gel chromatography in hexane/ethyl acetate (9:1)followed by preparative thin-layer chromatography on silica using thesame solvent mixture. After removal of the solvent and re-dissolving inhexane, the trans,cis-nepetalactone was crystallized on dry ice. GC-MSand NMR (¹H and ¹³C) analysis confirmed the identity of the crystallinematerial as trans,cis-nepetalactone. The ¹³C chemical shifts, comparedto the chemical shifts of Table 1, are shown in Table 5. TABLE 5 ¹³Cchemical shifts of the nepetalactone sample prepared in Example 4,compared to the chemical shifts of trans, cis-nepetalactone (fromTable 1) trans, cis- nepetalactone Sample Atom δ (ppm) δ (ppm) a 170.1170.3 b 135.9 136.0 c 120.4 120.5 d 37.3 37.5 e 49.1 49.3 f 32.1 32.2 g30.0 30.1 h 26.1 26.3 j 17.5 17.7 i 14.2 14.4

Hydrogenation of the trans,cis-nepetalactone thus prepared was carriedout in ethanol using ESCAT#142 catalyst (Englehart) at 50° C. for 4h.GC-MS and NMR (¹H and ¹³C) confirmed that the trans,cis-nepetalactonehad been quantitatively converted to the correspondingdihydronepetalactone stereoisomers, with one in significant excess. NMRanalysis of the major diastereomer: ¹H NMR (500 MHz, CDCl₃): d 0.97 (d,3H, J=6.28 Hz), 0.98 (d, 3H, J=6.94 Hz) d 1.24 (m, 2H), 1.74 (m, 1H),1.77 (m, 2H), 1.99 (m, 2H), 2.12 (dd, 1H, J=6.86 and 13.2 Hz), 2.51 (m,1H), 3.78 (tr, 1H, J=11.1 Hz), 4.33 (dd, 1H, J=5.73 and 11.32 Hz); ¹³C(500 MHz, CDCl₃): d 15.43, 18.09, 27.95, 30.81, 31.58, 35.70, 42.51,51.40, 76.18, 172.03. The ¹³C NMR spectrum indicated that this major 20diastereomer constituted ca. 93.7% of the preparation. Based on theobserved couplings for the methylene to the lactone oxygen, thestereogenic methine carbon bearing methyl group, the methyl group itselfand the bridgehead methine, it is concluded that the diastereomer ismost likely the(1S,9S,5R,6R)-5,9-dimethyl-3-oxabicyclo[4.3.0]nonan-2-one) of Formula 4.

The magnitude of the observed couplings are consistent with 10 dihedralangles between the protons on vicinal carbon atoms in the abovestructure according to the Karplus equation (ref. SpectrophotometricIdentification of Organic Compounds, 4th. edition, Robert M.Silverstein, G. Clayton Bassler and Terence C. Morill, 1981, page208-210).

Example 5 Repellency Testing of Dihydronepetalactones Prepared byHydrogenation of trans.cis-Nepetalactone

The dihydronepetalactone prepared according to Example 4, consistingpredominantly of1S,9S,5R,6R-5,9-dimethyl-3-oxabicylco[4.3.0]nonan-2-one; Formula 4, wastested for repellency against Aedes aegypti mosquitoes essentially asdescribed in Example 3. The experimental design is summarized in Table6, and all data presented is from five replicate experiments. TABLE 6Experimental Design Applied for Repellency Testing Purpose CompoundConcentration Untreated Control Isopropyl alcohol 100% Positive ControlIsopropyl alcohol with DEET 1.0% (w/v) Experimental Samples Isopropylalcohol with 1.0% (w/v) Dihydronepetalactone 0.5% (w/v) 0.2% (w/v)

Table 7 presents the effect of dihydronepetalactone concentration withrespect to the amount of time taken before the female A. aegyptiimosquitoes first probed each membrane. TABLE 7 Effect ofDihydronepetalactone Concentration on Mean Time to “First Probing”Repellent Concentration Mean Time (min) Isopropyl alcohol (untreatedcontrol) 8.0   1% DEET (positive control) 14.8   1% DHN 16.0 0.5% DHN10.4 0.2% DHN 8.4

Dihydronepetalactone at 1% concentration was found to discouragemosquito “first probing” for approximately 16 min, marginally betterthan (but statistically indistinguishable from) DEET at the sameconcentration, where mean time to first probe was 14.8 min. Lowerconcentrations of dihydronepetalactone (0.5% and 0.2% w/v) were found toinhibit first probing for an average of 10.4 and 8.4 min, respectively.

The distribution of landing density by female A. aegypti on membranestreated with dihydronepetalactones was analyzed over time, as showngraphically in FIG. 7. Dihydronepetalactone at 1.0% concentration wasfound to completely eliminate mosquito landings for 10 minutes, whileDEET (1% w/v) permitted mosquitoes to land by 8 min. Again, lowerconcentrations of dihydronepetalactone (0.5% and 0.2% w/v) were found toexhibit repellency (as compared to the untreated control), but at lowerlevels than the positive control (DEET at 1% (w/v)).

The total number of landings permitted on each membrane during thecourse of the experiments were determined, and the results aresummarized in Table 8. TABLE 8 Number of Landings Permitted According toRepellent and Concentration During 2 minute Observation PeriodsRepellent Concentration Mean Number of Landings Isopropyl alcohol(untreated 18.17 control)   1% DEET (positive control) 4.8   1% DHN 4.00.5% DHN 16.2 0.2% DHN 23.2

Again, this data indicates that 1% dihydronepetalactone exhibitedequivalent repellent activity to 1% DEET.

Cumulatively, this data show that at all three concentrations tested,treatment with dihydronepetalactone significantly reduced probing of themembranes over the course of the experiment (20 min) as compared to theisopropyl alcohol treatment. At all concentrations tested,dihydronepetalactones had effectiveness as a repellent of mosquitoes forthe entire observation period. There was also a direct relationshipbetween dihydronepetalactone concentration and the time elapsed for thefirst mosquito to land and begin probing the membrane. Overall, the dataindicate that the dihydronepetalactones derived from hydrogenation oftrans,cis-nepetalactone are an effective repellent, and equivalent toDEET in efficacy in these tests.

1. An insect repellent composition comprising1S,9S,5R,6R-5,9-dimethyl-3-oxabicyclo[4.3.0]nonan-2-one and one or morecarriers.
 2. The composition of claim 1 which comprises1S,9S,5R,6R-5,9-dimethyl-3-oxabicyclo[4.3.0]nonan-2-one in an amount offrom about 0.01% to about 50% by weight of the total weight of thecomposition.
 3. The composition of claim 1 further comprising one ormore adjuvants.
 4. The composition of claim 3 wherein the adjuvant isselected from one or more members of the group consisting of thickeners,buffering agents, chelating agents, preservatives, fragrances,antioxidants, gelling agents, stabilizers, surfactants, emollients,coloring agents, aloe vera, waxes, and therapeutically or cosmeticallyactive ingredients.
 5. The composition of claim 1 wherein the carrier isselected from the group consisting of water, silicone, petrolatum,lanolin, liquid hydrocarbons, agricultural spray oils, paraffin oil,tall oils, liquid terpene hydrocarbons and terpene alcohols, aliphaticand aromatic alcohols, esters, aldehydes, ketones, mineral oil, higheralcohols, finely divided organic and inorganic solid materials andmixtures thereof.
 6. The composition of claim 1 wherein the carriercomprises an aerosol composition adapted to disperse1S,9S,5R,6R-5,9-dimethyl-3-oxabicyclo[4.3.0]nonan-2-one into theatmosphere by means of a compressed gas.
 7. The composition of claim 1further comprising(9S,5R,1R,6R)-5,9-dimethyl-3-oxabicyclo[4.3.0]nonan-2-one.
 8. Thecomposition of claim 1 which is in the form of a cologne, a lotion, aspray, a cream, a gel, an ointment, a bath or shower gel, a foamproduct, makeup, a deodorant, shampoo, a hair lacquer or rinse, apersonal soap, an air freshener, a candle, a scented article, a fiber, asheet, cloth, paper, paint, ink, clay, wood, furniture, carpeting,sanitary goods, a plastic, or a polymer.
 9. An insect repellentcomposition that consists essentially of1S,9S,5R,6R-5,9-dimethyl-3-oxabicyclo[4.3.0]nonan-2-one,(9S,5R,1R,6R)-5,9-dimethyl-3-oxabicyclo[4.3.0]nonan-2-one and one ormore carriers.
 10. The composition of claim 9 that further consistsessentially of one or more adjuvants.
 11. An insect repellentcomposition comprising a dihydronepetalactone, or a mixture ofdihydronepetalactone stereoisomers, represented by the general formula:

and an essential oil selected from one or more members of the groupconsisting of almond bitter oil, anise oil, basil oil, bay oil, carawayoil, cardamom oil, cedar oil, celery oil, chamomile oil, cinnamon oil,citronella oil, clove oil, coriander oil, cumin oil, dill oil,eucalyptus oil, fennel oil, ginger oil, grapefruit oil, lemon oil, limeoil, mint oil, parsley oil, peppermint oil, pepper oil, rose oil,spearmint oil (menthol), sweet orange oil, thyme oil, turmeric oil, andoil of wintergreen.
 12. The composition of claim 11 wherein thedihydronepetalactone comprises one or both of1S,9S,5R,6R-5,9-dimethyl-3-oxabicyclo[4.3.0]nonan-2-one, and(9S,5R,1R,6R)-5,9-dimethyl-3-oxabicyclo[4.3.0]nonan-2-one.
 13. Thecomposition of claim 11 which comprises dihydronepetalactone in anamount of from about 0.01% to about 50% by weight of the total weight ofthe composition.
 14. The composition of claim 11, which comprises one ormore of the members of the group consisting of an adjuvant and acarrier.
 15. The composition of claim 14, wherein the adjuvant isselected from one or more members of the group consisting of thickeners,buffering agents, chelating agents, preservatives, fragrances,antioxidants, gelling agents, stabilizers, surfactants, emollients,coloring agents, aloe vera, waxes, and therapeutically or cosmeticallyactive ingredients.
 16. The composition of claim 14, wherein the carrieris selected from the group consisting of water, silicone, petrolatum,lanolin, liquid hydrocarbons, agricultural spray oils, paraffin oil,tall oils, liquid terpene hydrocarbons and terpene alcohols, aliphaticand aromatic alcohols, esters, aldehydes, ketones, mineral oil, higheralcohols, finely divided organic and inorganic solid materials andmixtures thereof
 17. The composition of claim 14, wherein the carriercomprises an aerosol composition adapted to disperse thedihydronepetalactone into the atmosphere by means of a compressed gas.18. The composition of claim 11, wherein the essential oil is selectedfrom one or more members of the group consisting of cinnamon oil,citronella oil, eucalyptus oil, ginger oil, grapefruit oil, lemon oil,lime oil, mint oil, peppermint oil, spearmint oil (menthol), sweetorange oil, and oil of wintergreen.
 19. The composition of claim 11which is in the form of a cologne, a lotion, a spray, a cream, a gel, anointment, a bath or shower gel, a foam product, makeup, a deodorant,shampoo, a hair lacquer or rinse, a personal soap, an air freshener, acandle, a scented article, a fiber, a sheet, cloth, paper, paint, ink,clay, wood, furniture, carpeting, sanitary goods, a plastic, or apolymer.
 20. A method of repelling insects comprising exposing theinsects to the composition of claim 1 or the composition of claim 11.21. The method of claim 20 wherein the composition is in the form of agas, liquid or solid.